The invention relates generally to the field of nucleic acid amplification, including methods of external controls that verify the absence or presence of specific target sequences during the polymerase chain reaction by detection of amplicons.
Nucleic acid amplification, and the polymerase chain reaction (PCR) in particular, is an important research tool, with applications in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like (Gilliland etal, (1990) Proc. Natl. Acad. Sci., 87:2725-2729; Bevan etal, (1992) PCR Methods and Applications, 1:222-228; Green etal, (1991) PCR Methods and Applications, 1:77-90). Descriptions of, and guidance for conducting, PCR are provided in extensive literature (Innis etal, (1989) in PCR Protocols, Academic Press, NY; McPherson etal, (1991) in PCR: A Practical Approach, Volume 1, Oxford University Press, Oxford, pp. 46, 199; McPherson etal, (1995) in PCR 2: A Practical Approach Oxford University Press, Oxford, pp. 7, 19).
PCR assays that discriminate and identify alleles are important to genotype DNA samples for specific mutations (Livak (1999) Genetic Analysis: Biomolecular Engineering, 14:143-49; Mein etal (2000) Genome Research, 10:330-43). PCR assays also provide relative quantification of gene expression (Gibson etal (1996) Genome Research, 6:995-1001; Heid etal (1996) Genome Research, 6:986-94; Yang etal (1993) Anal. Biochem. 208:110-16; Germer etal (1999) Genome Research, 9:72-78).
Fluorescence-based approaches to provide end-point or real-time measurements of PCR amplification products (amplicons) (Holland etal, (1991) Proc. Natl. Acad. Sci., 88:7276-80) have either employed intercalating dyes, e.g. ethidium bromide, to indicate the amount of double stranded DNA present (Gelfand etal, U.S. Pat. No. 5,210,015) or probes containing reporter-quencher pairs (xe2x80x9cTaqMan(copyright)xe2x80x9d, 5xe2x80x2 nuclease assay) that are cleaved during amplification to release a fluorescent signal proportional to the amount of double stranded DNA present (Livak etal, U.S. Pat. No. 5,538,848; Gelfand etal, U.S. Pat. No. 5,804,375).
Parallel control tests that confirm conditions for amplification of the target are desirable. PCR is often plagued by false positives due to template contamination from adjacent wells, pipetting errors, or aerosol transmission, especially in high density or high-throughput formats, such as 96-, 384-well, or higher density microtitre plate, or other array configurations. In addition, PCR suffers from false negative results when enzyme inhibitors are present in the target samples or when reagents are missing or degraded. Control amplification reactions are desirable for (i) normalization of quantification results, (ii) detection of amplification inhibitors in the target and other reagents, and (iii) to establish background signal levels. Positive amplification control tests for PCR give a detectable amplicon derived from a control template that is separate and distinct from the target. Detection of the positive control amplicon indicates that amplification is viable and operative within the reaction chamber. Positive amplification control tests which give no detectable product from the control components, indicate conditions within the reaction chamber that do not allow amplification, such as contaminants that inhibit PCR.
Internal control PCR is conducted in the same vessel, concurrently with PCR of the target sample polynucleotide (Coen, D. xe2x80x9cQuantification of DNAs by the Polymerase Chain Reaction Using an Internal Controlxe2x80x9d in The Polymerase Chain Reaction (1994) Mullis etal, Eds., Birkhauser, Boston, Mass., pp. 89-96; Coen, D. xe2x80x9cQuantitation of rare DNAs by polymerase chain reactionxe2x80x9d in Current Protocols in Molecular Biology (1990) Ausubel, etal, Eds. Greene Publ. Assoc. and Wiley-Interscience). Amplification of an internal control polynucleotide (ICP) with primers common to the target polynucleotide and the internal control polynucleotide, gives verification of true or false negatives, i.e. if target is not detected (Gibson etal (1996) Genome Research 6:995-1001). An ICP is often an endogenous region of the target polynucleotide sample, i.e. from the same source, genome, chromosome, gene, plasmid, or fragment as the target, thus normalizing for variation in the amount of target polynucleotide. An ICP may be an endogenous RNA or DNA sequence which is present in each experimental sample as isolated. An ICP may be an exogenous or foreign sequence of RNA or DNA which is spiked in to the target sample at a known concentration. The exogenous ICP may be an in vitro construct, used to distinguish true target negatives from PCR inhibition and normalize for differences in efficiency of sample extraction cDNA synthesis (Aoyagi, U.S. Pat. No. 5,952,202).
A pervasive difficulty with internal controls is keeping amplification of the control polynucleotide from interfering with target amplification or detection of the product (Reischl etal (1995) xe2x80x9cQuantitative PCRxe2x80x9d in Molecular Biotechnology, Vol. 3, Humana Press Inc., pp.55-71). Endogenous ICP are subject to amplification inhibitors and can therefore give a false negative signal. Endogenous ICP also may have priming sites for target primers and therefore give a false positive signal. Where endogenous ICP systems share one or more primers with the target, exhaustion of the shared primers may lead to inaccurate PCR quantification and limited dynamic range. In view of the limitations and deficiencies of conventional controls for the quantification and detection of nucleic acid amplification products, non-gel based, external control PCR methods that provide positive indications of amplification are desirable.
The present invention is directed towards novel methods, compositions and kits of reagents for detecting nucleic acid amplification of a single-stranded, external control, polynucleotide (ECP) concurrently with nucleic acid amplification of a known or unknown target polynucleotide.
In a first aspect, the invention provides a method for detecting a target polynucleotide sequence by amplifying a target polynucleotide with primer extension reagents in a first set of one or more vessels (FIG. 1A) and amplifying a single-stranded external control polynucleotide with the primer extension reagents in a second set of one or more vessels (FIG. 1B). The primer extension reagents include a forward primer, a reverse primer, one or more detectable probes, a polymerase, and one or more deoxynucleotide 5xe2x80x2-triphosphates (dNTP). The target polynucleotide, and the ECP and its complement contain sequences complementary to the primers and are amplified by the same forward and reverse primers during a PCR assay.
The forward primer and the detectable probe are adjacent or substantially adjacent when hybridized to the single-stranded external control polynucleotide, or its complement. Also, the reverse primer and the detectable probe are adjacent or substantially adjacent when hybridized to the single-stranded external control polynucleotide, or its complement. Signals, such as fluorescence, are then detected from the detectable probes.
In one embodiment, the detectable probes are self-quenching fluorescence probes (SQP), each comprising reporter dye and quencher moieties.
The primer extension reagent of the second set of vessels may include a first detectable probe and a second detectable probe. The sequence of the first probe differs from the second probe by one or more mismatches, insertions, or deletions. The signal from the first probe is resolvable from the signal of the second probe.
In another embodiment, a third set of one or more vessels includes a second single-stranded ECP and primer extension reagents. The sequence of the first single-stranded ECP differs from the second single-stranded ECP by one or more mismatches, insertions, or deletions. The sequence portion of the first single-stranded ECP complementary to a detectable probe differs by a single nucleotide from the sequence portion of the second single-stranded ECP complementary to the detectable probe.
In another aspect, the invention provides kits of reagents for nucleic acid amplification comprising: a single-stranded external control polynucleotide a forward primer, a reverse primer, a nucleic acid polymerase having 5xe2x80x2 nuclease activity, a detectable probe, one or more nucleotide 5xe2x80x2-triphosphates; and other primer extension reagents necessary for nucleic acid amplification. In one embodiment, the primers, self-quenching fluorescence probes, and primer extension reagents are conveniently dispensed as a kit of reagents.